Palkar Prajakta S, Kakhniashvili David G, Goodman Steven R, Mehendale Harihara M
Department of Toxicology, College of Pharmacy, University of Louisiana at Monroe, Monroe, LA 71209, USA.
Toxicol Appl Pharmacol. 2008 Aug 1;230(3):338-45. doi: 10.1016/j.taap.2008.03.005. Epub 2008 Mar 15.
Administration of a low priming dose of 2-butoxyethanol (BE, 500 mg/kg, p.o.) 7 days prior to a larger LD(90) dose (1500 mg BE/kg, p.o.) offers protection against the lethal dose-induced hemolysis and death in female Sprague Dawley rats because of prompt and efficient replacement of red blood cells (RBCs) with new resilient RBCs. The objective of the present work was to analyze the altered proteome of RBCs upon priming with BE in order to identify the potential anti-hemolytic survival proteins induced in the primed rat RBCs (P-RBCs) as opposed to vehicle-treated RBCs (V-RBCs). The RBCs from the two groups were fractionated into membrane and cytosolic fractions. The cytosolic fractions were further fractionated for proteomic analysis into 3 fractions. The fractions were labeled with Cy3 and Cy5 fluorescent dyes and subjected to 2-dimensional differential gel electrophoresis (DIGE) to analyze the protein profiles. Seven membrane and 8 cytosolic proteins were found to be significantly increased (> or =2.5 fold) in P-RBCs as compared to V-RBCs. The identified proteins can be classified into antioxidant, membrane skeleton, protein turnover, lipid raft, and energy metabolism components. Increased levels of the proteins from antioxidant and membrane skeleton groups were confirmed by Western blot analysis. The study provides the first report on protein profiling of rat RBCs as well as on alteration of the proteome upon exposure to a priming dose of hemotoxicant. Further studies are needed to prove the protective role of the identified proteins and will initiate the field of survival/protective/anti-hemolytic proteins in RBCs.
在给予较大的半数致死剂量（LD(90)，1500毫克/千克，口服）前7天，给予低剂量的引发剂量2-丁氧基乙醇（BE，500毫克/千克，口服），可保护雌性斯普拉格-道利大鼠免受致死剂量诱导的溶血和死亡，这是因为新的有弹性的红细胞能迅速有效地替代红细胞（RBC）。本研究的目的是分析用BE引发后红细胞蛋白质组的变化，以确定在引发的大鼠红细胞（P-RBC）中诱导产生的潜在抗溶血存活蛋白，与用赋形剂处理的红细胞（V-RBC）相对比。将两组的红细胞分离成膜组分和胞质组分。胞质组分进一步分离成3个组分用于蛋白质组分析。这些组分用Cy3和Cy5荧光染料标记，并进行二维差异凝胶电泳（DIGE）以分析蛋白质谱。与V-RBC相比，发现P-RBC中有7种膜蛋白和8种胞质蛋白显著增加（≥2.5倍）。鉴定出的蛋白质可分为抗氧化剂、膜骨架、蛋白质周转、脂筏和能量代谢成分。通过蛋白质印迹分析证实了抗氧化剂和膜骨架组蛋白质水平的增加。该研究首次报道了大鼠红细胞的蛋白质谱以及暴露于引发剂量的血液毒素后蛋白质组的变化。需要进一步研究以证明所鉴定蛋白质的保护作用，并将开启红细胞中存活/保护/抗溶血蛋白领域的研究。
